My long range goal, in the proposed project, is the elucidation of molecular mechanisms governing normal and pathologic expression of the genes for the major human red blood cell membrane skeleton protein spectrin. Alpha and beta spectrin mRNA levels, synthesis and degradation will be measured in normal red cell maturation. These parameters will also be investigated in certain human red blood cell diseases, inherited hemolytic anemias, associated with spectrin deficiency. I will isolate the alpha and beta spectrin gene promoters by molecular cloning. These will be analyzed by restriction mapping and nucleotide sequencing. Functional activity of the spectrin promoters will be studied in erythroid heterologous gene expression systems, using mouse erythroleukemia cells and the interleukin-3 dependent murine stem cell line FDC-Pmix. I will fuse the spectin gene promoters to chloramphenical acetyltransferase genes for ease in quantitation of their biological effects in the heterologous gene expression systems. Experimental mutagenesis of normal spectin promoters will allow detailed molecular dissection of the regions involved in red blood cell-specific gene expression. Promoters from any patients found to have deficient synthesis of spectrin mRNA will be isolated by molecular cloning. These will be investigated by mapping, sequencing, and functional assay in a heterologous gene expression system. It is hoped that these studies will lead to a greater understanding of gene regulation in red blood cell development, illuminate the molecular pathogenesis of inherited hemolytic anemias, and allow improved detection of the responsible genes in man.